- Email: [email protected]
Subchronic inhalation of particulate matter 10 coal dust induces atherosclerosis in the aorta of diabetic and nondiabetic rats
+
MODEL
Biomarkers and Genomic Medicine (2014) xx, 1e7
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.j-bgm.com
ORIGINAL ARTICLE
Subchronic inhalation of particulate matter 10 coal dust induces atherosclerosis in the aorta of diabetic and nondiabetic rats Bambang Setiawan a,*, Nia Kania a, Dian Nugrahenny b, Nurdiana Nurdiana b, Moch. Aris Widodo b a Research Center for Toxicology, Cancer, and Degenerative Disease, Department of Pathology, Ulin General Hospital, Medical Faculty Lambung Mangkurat University, Banjarmasin, South Kalimantan, Indonesia b Department of Pharmacology, Faculty of Medicine, Brawijaya University, Malang, East Java, Indonesia
Received 26 January 2014; received in revised form 5 March 2014; accepted 10 March 2014
KEYWORDS diabetes mellitus; endothelial damage; foam cells; oxidative stress; particulate matter 10
Abstract This study aimed to elucidate whether subchronic inhalation of particulate matter (PM10) coal dust induces atherosclerosis in diabetic rats. A total of 32 male Wistar rats, were randomly divided into eight groups including four nondiabetic groups and four groups of diabetic rats. These rats were exposed to doses of coal dust equal to 0 mg/m3, 6.25 mg/m3, 12.5 mg/m3, or 25 mg/m3 for 1 hour/day for 28 days. Plasma levels of lipid peroxides were determined as thiobarbituric acid reactive substance. The levels of circulating endothelial cells were analyzed histologically. Foam cells formation was analyzed in aorta and tail artery with Oil Red O staining. Analysis of variance test was used to compare all parameters. Nondiabetic rats exposed to coal dust had significantly increased oxidative stress compared to the control group (p < 0.05). Diabetic rats exposed to coal dust at dose of 25 mg/m3 had significantly increased oxidative stress compared to that of control diabetic rats (p < 0.05). The levels of endothelial damage were significantly increased in diabetic rats exposed to coal dust at doses of 6.25 mg/m3 and 12.5 mg/m3 (p < 0.05) compared to control diabetic rats. The foam cell counts were significantly increased in the aorta of nondiabetic rats exposed to coal dust at doses of 6.25 mg/m3 and 25 mg/m3 compared to control rats (p < 0.05), also in the tail artery at dose of 25 mg/m3. The foam cells counts were significantly increased in the aorta of diabetic rats exposed to coal dust at doses of 6.25 mg/m3 and 12.5 mg/m3 than that
* Corresponding author. Department of Medical Chemistry and Biochemistry Medical Faculty Lambung Mangkurat University, Jalan Veteran, Banjarmasin, South Kalimantan, Indonesia. E-mail address: [email protected] (B. Setiawan). http://dx.doi.org/10.1016/j.bgm.2014.03.002 2214-0247/Copyright ª 2014, Taiwan Genomic Medicine and Biomarker Society. Published by Elsevier Taiwan LLC. All rights reserved.
Please cite this article in press as: Setiawan B, et al., Subchronic inhalation of particulate matter 10 coal dust induces atherosclerosis in the aorta of diabetic and nondiabetic rats, Biomarkers and Genomic Medicine (2014), http://dx.doi.org/10.1016/j.bgm.2014.03.002
+
MODEL
2
B. Setiawan et al. in control diabetic rats (p < 0.05). Subchronic inhalation of PM10 coal dust induces atherosclerosis through oxidative stress and endothelial damage in aorta of nondiabetic and diabetic rats. Copyright ª 2014, Taiwan Genomic Medicine and Biomarker Society. Published by Elsevier Taiwan LLC. All rights reserved.
Introduction The World Coal Institute estimates that coal will remain a dominant energy source (i.e., remain at current production levels) due to the high price of oil.1 Recent studies have shown that coal dust induces respiratory as well as cardiovascular diseases. It was recently demonstrated that the population residing near the Appalachian coal mine facilities suffers from a higher rate of cardiovascular disease (e.g., coronary heart disease, heart attacks).2 Cardiovascular disease is the main complication of diabetes mellitus, whereas atherosclerosis is the main manifestation of diabetic microangiopathy.3,4 Previous study showed an association between particulate matter exposure and insulin resistance, cardiometabolic disorders, also increase in blood fasting glucose and HbA1c levels significantly.5,6 To date, no study has explored the effect of coal dust on diabetes mellitus vascular complications. Thus, the mechanism by which diabetes complications, especially atherosclerosis, are exacerbated by coal dust exposure is still unknown. Coal dust produces free radicals7 and increases oxidative stress in vitro8,9 and in vivo,10e13 which can lead to endothelial apoptosis14 as an initial step of atherogenesis. Assessing circulating endothelial cells (CECs), mature cells detached from the vascular intimal layer in response to a variety of insults, is a novel means of assessing endothelial damage, with increased counts of CECs being taken to imply vascular damage.15 Subsequently, endothelial damage provides macrophage cells with access to the subendothelial space. Moreover, low-density lipoprotein (LDL) oxidation in the subendothelial space induces the transformation of monocytes into foam cells.16 We hypothesize that the inhalation of coal dust induces the accumulation of particles in the vascular endothelium, which increases oxidative stress, endothelial damage, and the formation of foam cells in nondiabetic rats. In diabetic rats, we found a higher degree of oxidative stress, endothelial damage, and the formation of foam cells compared to nondiabetic rats. Thus, the main objective of this study was to determine whether coal dust exposure significantly increases atherosclerosis through oxidative stress, endothelial damage, and the formation of foam cells in nondiabetic and diabetic rats.
Material and methods Animals Thirty-two, male Wistar albino rats, aged 16 weeks, weighing 160e200 g were used for the present investigation.
They were housed in a clean wire cage and maintained under standard laboratory conditions (temperature 25 2 C with a 12-hour dark/light cycle). They were fed a standard pellet diet and received water ad libitum. The animals were acclimatized to laboratory conditions for 1 week prior to the experiment.
Coal dust preparation Coal dust was prepared according to our previous studies.17 In order to collect particulate matter 75 (PM75), subbituminous gross coal obtained from coal mining in South Kalimantan was pulverized using a Ball Mill, Ring Mill, and Raymond Mill in the Carsurin Coal Laboratories of Banjarmasin. Subsequently, we filtered PM75 using Mesh MicroSieve (BioDesign, USA) resulting in a coal dust <10 mm of diameter (PM10). PM10 coal dust was characterized by scanning electron microscope, X-ray fluorescence, and X-ray diffraction at the Physics and Central Laboratory, Faculty of Mathematics and Natural Science, University of Malang.
Induction of diabetes Diabetes was induced by a single intraperitoneal injection of a freshly buffered (0.1 M citrate, pH 4.5) solution of streptozotocin at dose of 60 mg/kg body weight. Tail vein blood was collected 72 hours after streptozotocin administration to determine the fasting blood glucose level. Only rats with fasting blood glucose over 250 mg/dL were considered diabetic and included in the experiments.18
Coal dust aerosolization A total of 32 male Wistar rats were randomly divided into eight groups including one nondiabetic group, three groups for coal dust exposure in nondiabetic rats, one diabetic group, and three groups for coal dust exposure in diabetic rats. Doses of coal dust exposure were 6.25 mg/m3, 12.5 mg/m3, and 25 mg/m3 for 1 hour/day for 28 days. The dose used for this exposure was according to coal dust concentration in the above ground coal mining area.19 Coal dust aerosolization was done by coal dust equipment that was designed and available in Department of Pharmacology, Faculty of Medicine, Brawijaya University of Malang. The principal of this equipment is to provide an ambient environment containing coal dust which then is inhaled by the rats. The airstream of this equipment is 1.5e2 L/minute, which mimics an environmental airstream.20
Please cite this article in press as: Setiawan B, et al., Subchronic inhalation of particulate matter 10 coal dust induces atherosclerosis in the aorta of diabetic and nondiabetic rats, Biomarkers and Genomic Medicine (2014), http://dx.doi.org/10.1016/j.bgm.2014.03.002
+
MODEL
Coal dust induces atherosclerosis in aorta
3
Tissue sampling
Statistical analysis
At the end of the treatment, the animals in all groups were anesthetized; the blood samples were then drawn by cardiac puncture and heparinized. Blood samples were centrifuged at a speed of 4000 g (4 minutes, 4 C) to obtain the plasma. The lungs, aorta, and tail artery were collected, weighed, and rinsed with physiological saline. All samples were stored at 80 C until analyzed.
Data are presented as mean standard deviation and differences between groups were analyzed using one-way analysis of variance (ANOVA) with SPSS version 15.0 (SPSS Inc., Chicago, IL, USA). A post hoc test was used if the result of ANOVA was significant. A p value < 0.05 was considered statistically significant.
Results Lipid peroxidation analysis Plasma levels of lipid peroxides were determined as thiobarbituric acid reactive substance (TBARS) according to the method of Ohkawa et al,21 based on the reaction of lipid peroxides with thiobarbituric acid (TBA) at 95 C. In the TBA test reaction, lipid peroxides and TBA react to form a pink pigment with an absorption maximum at 532 nm. The reaction was performed at pH 2e3 at 95 C for 15 minutes. The sample was mixed with 2.5 volumes of 10% (w/v) trichloroacetic acid to precipitate the protein. The precipitate was pelleted by centrifugation and an aliquot of supernatant was reacted with 0.67% TBA in a boiling water-bath for 15 minutes. After cooling, the absorbance was read at 532 nm. Arbitrary values obtained were compared with a series of standard solutions (1,1,3,3 tetramethoxypropane). Results were expressed as mM.
Circulating endothelial cells analysis Circulating endothelial cells were analyzed using a histology technique adapted from Hladovec and Rossman.22 A 2 mL blood sample was mixed with 0.2 mL sodium citrate 3.8% then centrifuged at 395 g 4 C for 20 minutes. The homogenate then added with 2 mL adenosine diphosphate as aggregator and centrifuged at 395 g 4 C for 20 minutes. The supernatant was collected then centrifuged at 395 g 4 C for 20 minutes. The pellet was resuspended in 0.1 mL NaCl 0.9% and transferred to an Improved Neubauer counting chamber. The cells were then visualized by light microscope and evaluated in two counting rooms. The number of CECs was expressed as cells/1.8 cm2.
Foam cells analysis Foam cells analysis was done in rat aorta and tail artery frozen section stained with Oil Red O. Foam cells, recognized as macrophages stained with Oil Red O, were visualized by light microscope with 40 magnification. The number of foam cells formed in each condition was calculated in triplicate manually and presented as percentage of total cells.
Coal dust characteristics To confirm the coal dust as PM10, scanning electron microscopy showed the highest diameter of particles to be <10 mm (in one dimension). Besides that, we also found particles in nanometer size called nanoparticles. The morphology of PM10 coal dust was small singlet particles linked together to form larger aggregate particles.23 X-ray diffraction showed 36.3% crystallinity with crystal size 177 nm, consisting of illite (potassium aluminum silicate hydroxide hydrate), viseite (calcium aluminum phosphate silicate hydroxyde), and cronstedtite (iron silicate hydroxyte). The inorganic components of the coal dust were iron (29.3 0.1%), silicon (29.0 0.2%), calcium (12.00 0.07%), aluminum (10 0.2%), titanium (6.31 0.19%), phosphorus (5.90 0.04%), and barium (1.00 0.09); and several inorganic minerals at concentration <1% including europium (0.70 0.00%), chromium (0.48 0.04%), nickel (0.41 0.00%), copper (0.34 0.02%), zinc (0.22 0.03%), vanadium (0.20 0.02%), and manganese (0.15 0.09%).20
Coal dust in the blood vessel Hematoxylineeosin staining of lung showed accumulation of coal dust in the blood vessel. Fig. 1 shows the structure of lung blood vessel (magnification 100e400); coal dust spread in erythrocytes indicating coal dust translocation into lung vascular system.
Effect of PM10 coal dust on oxidative stress Fig. 2 summarizes the levels of lipid peroxidation as an oxidative marker in the serum of control and experimental groups of rats. Nondiabetic rats exposed to coal dust had a significantly higher TBARS level than control rats (p < 0.05). The level of TBARS was significantly increased in diabetic rats compared to nondiabetic rats with or without exposure to coal dust (p < 0.05). The level of TBARS was significantly increased in diabetic rats exposed to coal dust at dose of 25 mg/m3 compared to the nonexposed diabetic rats (p < 0.05).
Effect of PM10 coal dust on endothelial damage Ethics This research had been approved by the Research Ethics Committee of the Faculty of Medicine, Brawijaya University, Malang, Indonesia.
The number of CECs is a marker of endothelial damage. The number of CECs in the control and experimental groups is shown in Fig. 3. The number of CECs was not significantly increased in nondiabetic rats exposed to coal dust
Please cite this article in press as: Setiawan B, et al., Subchronic inhalation of particulate matter 10 coal dust induces atherosclerosis in the aorta of diabetic and nondiabetic rats, Biomarkers and Genomic Medicine (2014), http://dx.doi.org/10.1016/j.bgm.2014.03.002
+
MODEL
4
B. Setiawan et al.
Figure 1 Coal dust accumulation in the lung blood vessels. The histologic structure of lung blood vessel (hematoxylineeosin staining, light microscope, magnification 100e400). (A) Coal dust in the lung blood vessel (black arrow) between red blood cells from nondiabetic rats; and nondiabetic rats exposed to coal dust at doses of (B) 6.25 mg/m3; (C) 12.5 mg/m3; and (D) 25 mg/m3. (E) Coal dust in nonexposed diabetic rats and diabetic rats exposed to coal dust at doses of (F) 6.25 mg/m3; (G) 12.5 mg/m3; and (H) 25 mg/m3. Coal dust in lung blood vessel indicates the translocation of particulate matter from alveoli into blood vessel.
compared to the control rats (p > 0.05). The level of CECs was significantly increased in diabetic rats compared to nondiabetic rats (p < 0.05). The level of CECs was significantly increased in diabetic rats exposed to coal dust at doses of 6.25 mg/m3 and 12.5 mg/m3 (p < 0.05) compared to that in diabetic rats not exposed to coal dust.
Effect of PM10 coal dust on foam cells counts in aorta The foam cells counts were significantly increased in the aorta of nondiabetic rats exposed to coal dust at doses of
Figure 2 Thiobarbituric acid reactive substance (TBARS) levels in nondiabetic and diabetic rats exposed to coal dust. Values are presented as mean standard deviation. a p < 0.05; in comparison with control group. b p < 0.05; in comparison with normal rats exposed to 6.25 mg/m3 coal dust. c p < 0.05; in comparison with normal rats exposed to 12.5 mg/m3 coal dust. d p < 0.05; in comparison with normal rats exposed to 25 mg/m3 coal dust. e p < 0.05; in comparison with diabetic rats. f p < 0.05; in comparison with diabetic rats exposed to 6.25 mg/m3 coal dust.
6.25 mg/m3 and 25 mg/m3 over that in the control rats (p < 0.05). Foam cells counts were significantly increased in aorta of diabetic rats exposed to coal dust at doses of 6.25 mg/m3 and 12.5 mg/m3 compared to control diabetic rats (p < 0.05), as shown in Fig. 4.
Effect of PM10 coal dust on foam cells counts in tail artery Foam cells count was significantly increased in tail artery of nondiabetic rats exposed to coal dust at dose of 25 mg/m3 compared to control rats (p < 0.05). The foam cells count was not significantly different in the tail artery of diabetic
Figure 3 Circulating endothelial cells (CECs) numbers in nondiabetic and diabetic rats exposed to coal dust. Values are presented as mean standard deviation. a p < 0.05; in comparison with control group. b p < 0.05; in comparison with normal rats exposed to 6.25 mg/m3 coal dust. c p < 0.05; in comparison with normal rats exposed to 12.5 mg/m3 coal dust. d p < 0.05; in comparison with diabetic rats.
Please cite this article in press as: Setiawan B, et al., Subchronic inhalation of particulate matter 10 coal dust induces atherosclerosis in the aorta of diabetic and nondiabetic rats, Biomarkers and Genomic Medicine (2014), http://dx.doi.org/10.1016/j.bgm.2014.03.002
+
MODEL
Coal dust induces atherosclerosis in aorta
Figure 4 Foam cells counts in aorta (blue bar) and tail artery (red bar) of nondiabetic and diabetic rats exposed to coal dust. Values are presented as mean standard deviation. a p < 0.05; in comparison with control group. b p < 0.05; in comparison with normal rats exposed to 6.25 mg/m3 coal dust. c p < 0.05; in comparison with normal rats exposed to 12.5 mg/m3 coal dust. d p < 0.05; in comparison with normal rats exposed to 25 mg/m3 coal dust. e p < 0.05; in comparison with diabetic rats. f p < 0.05; in comparison with diabetic rats exposed to 6.25 mg/m3 coal dust. g p < 0.05; in comparison with diabetic rats exposed to 12.5 mg/m3 coal dust.
rats exposed to coal dust compared to that in nonexposed diabetic rats (p > 0.05), as shown in Fig. 4.
Discussion Prior to exposure, we characterized coal dust using scanning electron microscopy, which demonstrated a diameter <10 mm at least in one dimension, in morphology of singlet or aggregate particles, with crystal size 177 nm. We arrived at two main findings. The first finding concerns the accumulation of PM10 coal dust in pulmonary blood vessel. The second is the effect of PM10 coal dust on foam cell formation as a vascular complication of diabetes mellitus. The accumulation of coal dust in lung blood vessel indicates the potential translocation of the dust from the alveolar into the circulatory system. The mechanism of translocation involves the increase in the lung epithelium permeability induced by oxidative stress. Furthermore, this process opens up the access of particles into the interstitial compartment and they continue to pass into the endothelial cells and the blood, and then they are circulated to several organs.24 Coal dust in nanoparticles is able to translocate from the lungs into the circulatory system directly. Although only very small doses of coal dust nanoparticles enter the circulation (1%), the effect in the targeted organs is caused by large surface particles and the retention time.25 TBARS is the product of lipid peroxidation as an oxidative stress marker. We found an increase in oxidative stress in all of the groups, when comparing them with the control group of nondiabetic rats. Oxidative stress in diabetic rats with or without coal dust inhalation was significantly higher than that in the nondiabetic rats. In nondiabetic rats, the inhalation of coal dust increased oxidative stress in a dose dependent manner compared to the control group (p < 0.05). In diabetic rats, coal dust inhalation significantly increased oxidative stress when compared to the control group at doses of 12.5 mg/m3 and 25 mg/m3 (p < 0.05). The coal dust used in this study contained
5 several metal redox properties, such as iron (29.3 0.1%), titanium (6.31 0.19%), chromium (0.48 0.04%), nickel (0.41 0.00%), vanadium (0.20 0.02%), and manganese (0.15 0.09%). This active metal content induces lipid peroxidation by a mechanism that is both hydroxyl radical dependent and nonhydroxyl radical dependent.26,27 In addition, hyperglycemia in diabetes mellitus also induces oxidative stress via several mechanisms, such as nonenzymatic glycation, the polyol pathway, and glucose autooxidation.28 There is accumulation of oxidative stress in diabetic rats with the inhalation of coal dust. CECs are mature cells detached from the intimal monolayer of the vascular wall. The count of these cells circulating in the peripheral blood is a biomarker of vascular damage. Several mechanisms can be related to endothelial detachment, including apoptosis, mechanical damage of cells, and protein lysis of the subendothelial matrix.29,30 Depending on the stress level, a severe degree of oxidative stress induces apoptosis in endothelial cells.14 We found a significant increase in CECs in diabetic groups compared to nondiabetic groups without coal dust exposure. This finding indicates that hyperglycemia increases endothelial damage and detachment. Coal dust increases endothelial damage in diabetic rats (at doses of 6.25 mg/m3 and 12.5 mg/m3) over that in control diabetic rats. This finding indicates that active metals in coal dust can damage endothelial cells may be due to apoptosis, mechanical damage of cells, protein lysis of the subendothelial matrix, or oxidative stress. At dose of 25 mg/m3, coal dust decreases circulating endothelial cells in diabetic rats to reach the level of control diabetic rats. This finding indicates that coal dust at this dose inhibits endothelial detachment in diabetic rats. The mechanism may be due to an inhibition of aggressive factor in diabetic rats. Free radicals from coal dust will react with the free radicals from hyperglycemia to induce the termination (neutralization) reaction. Our previous studies showed that coal dust exposure tended to decrease endothelial cells damage in rats fed a high-cholesterol diet.31 Foam cells are a histologic marker of early atherosclerotic lesions. In this study, we assessed the foam cell formations in large and small arteries of rats. In nondiabetic rats, coal dust inhalation significantly increased the foam cells count in the aorta at doses of 6.25 mg/m3 and 25 mg/m3 compared to the control group (p < 0.05). Moreover, when nondiabetic rats are exposed to 25 mg/m3 coal dust, foam cells increased in the tail artery compared to the control and lower exposure dose. These findings indicate that active metals in coal dust initiate an early lesion of atherosclerosis in large and small vessels of nondiabetic rats. Increased oxidation of LDL-cholesterol is a key feature of foam cells, and transition metals can increase both direct LDL-cholesterol oxidation and LDL oxidation by monocytes.32,33 Also, previous study have proved that coal dust increases systemic inflammation.13 In diabetic rats, inhalation of coal dust significantly increased foam cells counts in aorta at doses of 6.25 mg/m3 and 12.5 mg/m3 compared to the control group (p < 0.05), but this did not reach statistically significance in the small artery. Actually, the concentration of collagen and elastin in the aorta is inversely related to that in the tail artery in rats. Collagen is lower in the aorta, but higher in the tail
Please cite this article in press as: Setiawan B, et al., Subchronic inhalation of particulate matter 10 coal dust induces atherosclerosis in the aorta of diabetic and nondiabetic rats, Biomarkers and Genomic Medicine (2014), http://dx.doi.org/10.1016/j.bgm.2014.03.002
+
MODEL
6 artery.34 Collagen induces progression of atherosclerotic lesion by serving as a depot of proatherogenic molecules.35 Elastin is an extracellular protein with lower turnover ratesdalmost 40 years.36 The total alkaline-insoluble aorta elastin content is significantly reduced in diabetic compared to control rats.37 The combination of collagen and elastin content in the aorta, oxidative stress level related to coal dust exposure (at doses of 6.25 mg/m3 and 12.5 mg/m3), and hyperglycemia, may be the reason for proatherogenic molecules deposition forming foam cells in the aorta of diabetic rats.38 At the highest dose, coal dust inhibited foam cells formation in the aorta or tail artery. Our previous study showed that coal dust decreases cholesterol and LDL-cholesterol levels at an exposure dose of 25 mg/m3, and this effect may contribute to this finding.39 In conclusion, our study found that subchronic inhalation of PM10 coal dust induces atherosclerosis through oxidative stress and endothelial damage in the aorta of nondiabetic and diabetic rats.
Conflicts of interest The authors declare that there is no conflicts of interests regarding the publication of this article.
Acknowledgments The authors are grateful to PT. Carsurin Banjarmasin, South Kalimantan for providing coal dust in diameter <70 mm. The authors also thank all technician in the Department of Pharmacology and Laboratory of Biomedicine (Faculty of Medicine, Brawijaya University, Malang, East Java, Indonesia) for helping in the procedure of coal dust exposure and analyses of the biological marker.
References 1. Huang X. Iron, oxidative stress, and cell signalling in the pathogenesis of coal worker’s pneumoconiosis, silicosis, and asbestosis. Am J Biomed Sci. 2001;3:95e106. 2. Hendryx M, Zullig KJ. Higher coronary artery disease and heart attack morbidity in Appalachian coal mining regions. Preventive Med. 2009;49:355e359. 3. Bonnefont-Rousselot D. Glucose and reactive oxygen species. Curr Opinion Clin Nutr Metab Care. 2002;5:561e568. 4. Wu T, McGrath KCY, Death AK. Cardiovascular disease in diabetic nephropathy patients: cell adhesion molecules as potential markers. Vasc Health Risk Manag. 2005;1:309e316. 5. Rajagopalan S, Brook RD. Air pollution and type 2 of diabetes. Diabetes. 2012;61:3037e3045. 6. Chuang KJ, Yan YH, Chiu SY, et al. Long-term air pollution exposure and risk factors for cardiovascular diseases among the elderly in Taiwan. Occup Environ Med. 2011;68:64e68. 7. Dalal NS, Newman J, Pack D, et al. Hydroxyl radical generation by coal mine dust: possible implication to coal workers pneumoconiosis. Free Radic Biol Med. 1995;18:1e20. 8. Zhang Q, Dai J, Ali A, et al. Roles of bioavailable iron and calcium in coal dust-induced oxidative stress: Possible implications in coal workers’ lung disease. Free Rad Res. 2002;36: 285e294.
B. Setiawan et al. 9. Zhang Q, Huang X. Induction of ferritin and lipid peroxidation by coal samples with different prevalence of coal workers’ pneumoconiosis: role of iron in the coals. Am J Ind Med. 2002; 42:171e179. 10. Armutcu F, Gun BD, Altin R, et al. Examination of lung toxicity, oxidant/antioxidant status and effect of erdosteine in rats kept in coal mine ambience. Environ Toxicol Pharmacol. 2007; 24:106e113. 11. Pinho RA, Silveira PCL, Piazza M, et al. Regular physical exercises decrease the oxidant pulmonary stress in rats after acute exposure to mineral coal. Rev Bras Med Esporte. 2004;12: 71ee74e. 12. Avila Ju ´nior SA, Possamai FP, Budni P, et al. Occupational airborne contamination in South Brazil: oxidative stress detected in blood of coal miners. Ecotoxicol. 2009;18:1150e1157. 13. Ulker OC, Yucesoy B, Demir O, et al. Serum and BAL cytokine and antioxidant enzyme level at different stages of pneumoconiosis in coal workers. Hum Exp Toxicol. 2008;27:871e877. 14. Aoki M, Nata T, Morishita R, et al. Endothelial apoptosis induced oxidative stress through activation of NFkB: antiapoptotic effect of antioxidant agents on endothelial cells. Hypertension. 2001;38:48e55. 15. Boos CK, Lip GYH, Blann AD. Circulating endothelial cells in cardiovascular disease. J Am Col Cardiol. 2006;48:1538e1547. 16. Lane HA, Smith JC, Davies SJ. Noninvasive assessment of preclinical atherosclerosis. Vasc Health Risk Manag. 2006;2: 19e36. 17. Kania N, Mayangsari E, Setiawan B, et al. The effects of Eucheuma cottonii on signaling pathway inducing mucin synthesis in rats lungs chronically exposed to particulate matter 10 (PM10) coal dust. J Toxicol. 2013;2013:528146. 18. Cheng D, Liang B, Li Y. Antihyperglycemic effect of Ginkgo biloba extract in streptozotocin-induced diabetes in rats. BioMed Res Int. 2013;2013:162724. 19. Gurel A, Armutcu F, Damatoglu S, et al. Evaluation of erythrocyte Naþ, Kþ, -ATPase and superoxide dismutase activities and malondialdehyde level alteration in coal miners. Eur J Gen Med. 2004;1:22e28. 20. Noor Z, Setiawan B. Subchronic inhaled particulate matter coal dust changes bone mesostructure, mineral element and turn over markers in rats. J Exp Integr Med. 2013;3:153e158. 21. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95: 351e358. 22. Hladovec B, Rossman P. Circulating endothelial cell isolated together with platelets and experimental modification of their counts in rats. Thrombos Res. 1973;3:665e674. 23. Kania N, Setiawan B, Widjajanto E, et al. Peroxidative index as novel marker of hydrogen peroxide involvement in lipid peroxidation from coal dust exposure. Oxid Antioxid Med Sci. 2012;1:209e215. 24. Donaldson K, Stone V, Seaton A, et al. Ambient particle inhalation and the cardiovascular system: potential mechanisms. Environ Health Perspect. 2001;109:523e527. 25. Delfino RJ, Staimer N, Vaziri ND. Air pollution and circulating biomarkers of oxidative stress. Air Quality Atmos Health. 2011; 4:37e52. 26. Broeckaert F, Buchet JP, Delos M, et al. Coal fly ash- and copper smelter dust-induced modulation of ex vivo production of tumor necrosis factor-alpha by murine macrophages: effects of metals and overload. J Toxicol Environ. 1999;56:343e360. 27. Valko M, Rhode CJ, Moncol J, et al. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact. 2006;160:1e40. 28. Mokini S, Marcovecchio ML, Chiarelli F. Molecular pathology of oxidative stress in diabetic angiopathy: role of mitochondrial and cellular pathways. Diabetes Res Clin Pract. 2010;87: 313e321.
Please cite this article in press as: Setiawan B, et al., Subchronic inhalation of particulate matter 10 coal dust induces atherosclerosis in the aorta of diabetic and nondiabetic rats, Biomarkers and Genomic Medicine (2014), http://dx.doi.org/10.1016/j.bgm.2014.03.002
+
MODEL
Coal dust induces atherosclerosis in aorta 29. Kondo S, Ueno H, Hashimoto J, et al. Circulating endothelial cells and other angiogenesis factors in pancreatic carcinoma patients receiving gemcitabine chemotherapy. BMC Cancer. 2012;12:268. 30. Elshal M, Abdelaziz A, Abbas A, et al. Quantification of circulating endothelial cells in peripheral blood of systemic lupus erythematosus patients: a simple and reproducible method of assesing endothelial injury and repair. Nephrol Dial Transplant. 2009;24:1495e1499. 31. Setiawan B, Darsuni A, Muttaqien F, et al. The effects of combined particulate matter 10 coal dust exposure and highcholesterol diet on lipid profiles, endothelial damage, and hematopoietic stem cells in rats. J Exp Integr Med. 2013;3: 219e223. 32. Xing X, Baffic J, Sparrow CP. LDL oxidation by activated monocytes: characterization of oxidized LDL and requirement for transition metals ions. J Lipid Res. 1998;39:2201e2208. 33. Retsky KL, Chen K, Zeind J, et al. Inhibition of copper-induced LDL oxidation by vitamin C is associated with decreased
7
34. 35. 36.
37.
38.
39.
copper-binding to LDL and 2-oxo-histidine formation. Free Rad Biol Med. 1999;26:90e98. Cox RH. Time course of artery wall changes with DOCA plus salt hypertension in the rat. Hypertension. 1982;4:27e38. Rekhter MD. Collagen synthesis in atherosclerosis: too much and not enough. Cardiovas Res. 1999;41:376e384. Yamamoto Y, Sakata N, Meng J, et al. Possible involvement of glycoxidation and lipid peroxidation of elastin in atherogenesis in haemodialysis patients. Nephrol Dial Transplant. 2002;17: 630e636. Kwan CY, Wang RR, Beazley JS, et al. Alterations of elastin and elastase-like activities in aortae of diabetic rats. Biochim Biophys Acta. 1988;967:322e325. Francis AA, Pierce GN. An integrated approach for the mechanisms responsible for atherosclerotic plaque regression. Exp Clin Cardiol. 2011;16:77e86. Setiawan B, Darsuni A, Muttaqien F, et al. Cholesterol lowering effect of subchronic inhalation particulate matter 10 coal dust on rats. Med-Sci. 2013;2:500e511.
Please cite this article in press as: Setiawan B, et al., Subchronic inhalation of particulate matter 10 coal dust induces atherosclerosis in the aorta of diabetic and nondiabetic rats, Biomarkers and Genomic Medicine (2014), http://dx.doi.org/10.1016/j.bgm.2014.03.002
MODEL
Biomarkers and Genomic Medicine (2014) xx, 1e7
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.j-bgm.com
ORIGINAL ARTICLE
Subchronic inhalation of particulate matter 10 coal dust induces atherosclerosis in the aorta of diabetic and nondiabetic rats Bambang Setiawan a,*, Nia Kania a, Dian Nugrahenny b, Nurdiana Nurdiana b, Moch. Aris Widodo b a Research Center for Toxicology, Cancer, and Degenerative Disease, Department of Pathology, Ulin General Hospital, Medical Faculty Lambung Mangkurat University, Banjarmasin, South Kalimantan, Indonesia b Department of Pharmacology, Faculty of Medicine, Brawijaya University, Malang, East Java, Indonesia
Received 26 January 2014; received in revised form 5 March 2014; accepted 10 March 2014
KEYWORDS diabetes mellitus; endothelial damage; foam cells; oxidative stress; particulate matter 10
Abstract This study aimed to elucidate whether subchronic inhalation of particulate matter (PM10) coal dust induces atherosclerosis in diabetic rats. A total of 32 male Wistar rats, were randomly divided into eight groups including four nondiabetic groups and four groups of diabetic rats. These rats were exposed to doses of coal dust equal to 0 mg/m3, 6.25 mg/m3, 12.5 mg/m3, or 25 mg/m3 for 1 hour/day for 28 days. Plasma levels of lipid peroxides were determined as thiobarbituric acid reactive substance. The levels of circulating endothelial cells were analyzed histologically. Foam cells formation was analyzed in aorta and tail artery with Oil Red O staining. Analysis of variance test was used to compare all parameters. Nondiabetic rats exposed to coal dust had significantly increased oxidative stress compared to the control group (p < 0.05). Diabetic rats exposed to coal dust at dose of 25 mg/m3 had significantly increased oxidative stress compared to that of control diabetic rats (p < 0.05). The levels of endothelial damage were significantly increased in diabetic rats exposed to coal dust at doses of 6.25 mg/m3 and 12.5 mg/m3 (p < 0.05) compared to control diabetic rats. The foam cell counts were significantly increased in the aorta of nondiabetic rats exposed to coal dust at doses of 6.25 mg/m3 and 25 mg/m3 compared to control rats (p < 0.05), also in the tail artery at dose of 25 mg/m3. The foam cells counts were significantly increased in the aorta of diabetic rats exposed to coal dust at doses of 6.25 mg/m3 and 12.5 mg/m3 than that
* Corresponding author. Department of Medical Chemistry and Biochemistry Medical Faculty Lambung Mangkurat University, Jalan Veteran, Banjarmasin, South Kalimantan, Indonesia. E-mail address: [email protected] (B. Setiawan). http://dx.doi.org/10.1016/j.bgm.2014.03.002 2214-0247/Copyright ª 2014, Taiwan Genomic Medicine and Biomarker Society. Published by Elsevier Taiwan LLC. All rights reserved.
Please cite this article in press as: Setiawan B, et al., Subchronic inhalation of particulate matter 10 coal dust induces atherosclerosis in the aorta of diabetic and nondiabetic rats, Biomarkers and Genomic Medicine (2014), http://dx.doi.org/10.1016/j.bgm.2014.03.002
+
MODEL
2
B. Setiawan et al. in control diabetic rats (p < 0.05). Subchronic inhalation of PM10 coal dust induces atherosclerosis through oxidative stress and endothelial damage in aorta of nondiabetic and diabetic rats. Copyright ª 2014, Taiwan Genomic Medicine and Biomarker Society. Published by Elsevier Taiwan LLC. All rights reserved.
Introduction The World Coal Institute estimates that coal will remain a dominant energy source (i.e., remain at current production levels) due to the high price of oil.1 Recent studies have shown that coal dust induces respiratory as well as cardiovascular diseases. It was recently demonstrated that the population residing near the Appalachian coal mine facilities suffers from a higher rate of cardiovascular disease (e.g., coronary heart disease, heart attacks).2 Cardiovascular disease is the main complication of diabetes mellitus, whereas atherosclerosis is the main manifestation of diabetic microangiopathy.3,4 Previous study showed an association between particulate matter exposure and insulin resistance, cardiometabolic disorders, also increase in blood fasting glucose and HbA1c levels significantly.5,6 To date, no study has explored the effect of coal dust on diabetes mellitus vascular complications. Thus, the mechanism by which diabetes complications, especially atherosclerosis, are exacerbated by coal dust exposure is still unknown. Coal dust produces free radicals7 and increases oxidative stress in vitro8,9 and in vivo,10e13 which can lead to endothelial apoptosis14 as an initial step of atherogenesis. Assessing circulating endothelial cells (CECs), mature cells detached from the vascular intimal layer in response to a variety of insults, is a novel means of assessing endothelial damage, with increased counts of CECs being taken to imply vascular damage.15 Subsequently, endothelial damage provides macrophage cells with access to the subendothelial space. Moreover, low-density lipoprotein (LDL) oxidation in the subendothelial space induces the transformation of monocytes into foam cells.16 We hypothesize that the inhalation of coal dust induces the accumulation of particles in the vascular endothelium, which increases oxidative stress, endothelial damage, and the formation of foam cells in nondiabetic rats. In diabetic rats, we found a higher degree of oxidative stress, endothelial damage, and the formation of foam cells compared to nondiabetic rats. Thus, the main objective of this study was to determine whether coal dust exposure significantly increases atherosclerosis through oxidative stress, endothelial damage, and the formation of foam cells in nondiabetic and diabetic rats.
Material and methods Animals Thirty-two, male Wistar albino rats, aged 16 weeks, weighing 160e200 g were used for the present investigation.
They were housed in a clean wire cage and maintained under standard laboratory conditions (temperature 25 2 C with a 12-hour dark/light cycle). They were fed a standard pellet diet and received water ad libitum. The animals were acclimatized to laboratory conditions for 1 week prior to the experiment.
Coal dust preparation Coal dust was prepared according to our previous studies.17 In order to collect particulate matter 75 (PM75), subbituminous gross coal obtained from coal mining in South Kalimantan was pulverized using a Ball Mill, Ring Mill, and Raymond Mill in the Carsurin Coal Laboratories of Banjarmasin. Subsequently, we filtered PM75 using Mesh MicroSieve (BioDesign, USA) resulting in a coal dust <10 mm of diameter (PM10). PM10 coal dust was characterized by scanning electron microscope, X-ray fluorescence, and X-ray diffraction at the Physics and Central Laboratory, Faculty of Mathematics and Natural Science, University of Malang.
Induction of diabetes Diabetes was induced by a single intraperitoneal injection of a freshly buffered (0.1 M citrate, pH 4.5) solution of streptozotocin at dose of 60 mg/kg body weight. Tail vein blood was collected 72 hours after streptozotocin administration to determine the fasting blood glucose level. Only rats with fasting blood glucose over 250 mg/dL were considered diabetic and included in the experiments.18
Coal dust aerosolization A total of 32 male Wistar rats were randomly divided into eight groups including one nondiabetic group, three groups for coal dust exposure in nondiabetic rats, one diabetic group, and three groups for coal dust exposure in diabetic rats. Doses of coal dust exposure were 6.25 mg/m3, 12.5 mg/m3, and 25 mg/m3 for 1 hour/day for 28 days. The dose used for this exposure was according to coal dust concentration in the above ground coal mining area.19 Coal dust aerosolization was done by coal dust equipment that was designed and available in Department of Pharmacology, Faculty of Medicine, Brawijaya University of Malang. The principal of this equipment is to provide an ambient environment containing coal dust which then is inhaled by the rats. The airstream of this equipment is 1.5e2 L/minute, which mimics an environmental airstream.20
Please cite this article in press as: Setiawan B, et al., Subchronic inhalation of particulate matter 10 coal dust induces atherosclerosis in the aorta of diabetic and nondiabetic rats, Biomarkers and Genomic Medicine (2014), http://dx.doi.org/10.1016/j.bgm.2014.03.002
+
MODEL
Coal dust induces atherosclerosis in aorta
3
Tissue sampling
Statistical analysis
At the end of the treatment, the animals in all groups were anesthetized; the blood samples were then drawn by cardiac puncture and heparinized. Blood samples were centrifuged at a speed of 4000 g (4 minutes, 4 C) to obtain the plasma. The lungs, aorta, and tail artery were collected, weighed, and rinsed with physiological saline. All samples were stored at 80 C until analyzed.
Data are presented as mean standard deviation and differences between groups were analyzed using one-way analysis of variance (ANOVA) with SPSS version 15.0 (SPSS Inc., Chicago, IL, USA). A post hoc test was used if the result of ANOVA was significant. A p value < 0.05 was considered statistically significant.
Results Lipid peroxidation analysis Plasma levels of lipid peroxides were determined as thiobarbituric acid reactive substance (TBARS) according to the method of Ohkawa et al,21 based on the reaction of lipid peroxides with thiobarbituric acid (TBA) at 95 C. In the TBA test reaction, lipid peroxides and TBA react to form a pink pigment with an absorption maximum at 532 nm. The reaction was performed at pH 2e3 at 95 C for 15 minutes. The sample was mixed with 2.5 volumes of 10% (w/v) trichloroacetic acid to precipitate the protein. The precipitate was pelleted by centrifugation and an aliquot of supernatant was reacted with 0.67% TBA in a boiling water-bath for 15 minutes. After cooling, the absorbance was read at 532 nm. Arbitrary values obtained were compared with a series of standard solutions (1,1,3,3 tetramethoxypropane). Results were expressed as mM.
Circulating endothelial cells analysis Circulating endothelial cells were analyzed using a histology technique adapted from Hladovec and Rossman.22 A 2 mL blood sample was mixed with 0.2 mL sodium citrate 3.8% then centrifuged at 395 g 4 C for 20 minutes. The homogenate then added with 2 mL adenosine diphosphate as aggregator and centrifuged at 395 g 4 C for 20 minutes. The supernatant was collected then centrifuged at 395 g 4 C for 20 minutes. The pellet was resuspended in 0.1 mL NaCl 0.9% and transferred to an Improved Neubauer counting chamber. The cells were then visualized by light microscope and evaluated in two counting rooms. The number of CECs was expressed as cells/1.8 cm2.
Foam cells analysis Foam cells analysis was done in rat aorta and tail artery frozen section stained with Oil Red O. Foam cells, recognized as macrophages stained with Oil Red O, were visualized by light microscope with 40 magnification. The number of foam cells formed in each condition was calculated in triplicate manually and presented as percentage of total cells.
Coal dust characteristics To confirm the coal dust as PM10, scanning electron microscopy showed the highest diameter of particles to be <10 mm (in one dimension). Besides that, we also found particles in nanometer size called nanoparticles. The morphology of PM10 coal dust was small singlet particles linked together to form larger aggregate particles.23 X-ray diffraction showed 36.3% crystallinity with crystal size 177 nm, consisting of illite (potassium aluminum silicate hydroxide hydrate), viseite (calcium aluminum phosphate silicate hydroxyde), and cronstedtite (iron silicate hydroxyte). The inorganic components of the coal dust were iron (29.3 0.1%), silicon (29.0 0.2%), calcium (12.00 0.07%), aluminum (10 0.2%), titanium (6.31 0.19%), phosphorus (5.90 0.04%), and barium (1.00 0.09); and several inorganic minerals at concentration <1% including europium (0.70 0.00%), chromium (0.48 0.04%), nickel (0.41 0.00%), copper (0.34 0.02%), zinc (0.22 0.03%), vanadium (0.20 0.02%), and manganese (0.15 0.09%).20
Coal dust in the blood vessel Hematoxylineeosin staining of lung showed accumulation of coal dust in the blood vessel. Fig. 1 shows the structure of lung blood vessel (magnification 100e400); coal dust spread in erythrocytes indicating coal dust translocation into lung vascular system.
Effect of PM10 coal dust on oxidative stress Fig. 2 summarizes the levels of lipid peroxidation as an oxidative marker in the serum of control and experimental groups of rats. Nondiabetic rats exposed to coal dust had a significantly higher TBARS level than control rats (p < 0.05). The level of TBARS was significantly increased in diabetic rats compared to nondiabetic rats with or without exposure to coal dust (p < 0.05). The level of TBARS was significantly increased in diabetic rats exposed to coal dust at dose of 25 mg/m3 compared to the nonexposed diabetic rats (p < 0.05).
Effect of PM10 coal dust on endothelial damage Ethics This research had been approved by the Research Ethics Committee of the Faculty of Medicine, Brawijaya University, Malang, Indonesia.
The number of CECs is a marker of endothelial damage. The number of CECs in the control and experimental groups is shown in Fig. 3. The number of CECs was not significantly increased in nondiabetic rats exposed to coal dust
Please cite this article in press as: Setiawan B, et al., Subchronic inhalation of particulate matter 10 coal dust induces atherosclerosis in the aorta of diabetic and nondiabetic rats, Biomarkers and Genomic Medicine (2014), http://dx.doi.org/10.1016/j.bgm.2014.03.002
+
MODEL
4
B. Setiawan et al.
Figure 1 Coal dust accumulation in the lung blood vessels. The histologic structure of lung blood vessel (hematoxylineeosin staining, light microscope, magnification 100e400). (A) Coal dust in the lung blood vessel (black arrow) between red blood cells from nondiabetic rats; and nondiabetic rats exposed to coal dust at doses of (B) 6.25 mg/m3; (C) 12.5 mg/m3; and (D) 25 mg/m3. (E) Coal dust in nonexposed diabetic rats and diabetic rats exposed to coal dust at doses of (F) 6.25 mg/m3; (G) 12.5 mg/m3; and (H) 25 mg/m3. Coal dust in lung blood vessel indicates the translocation of particulate matter from alveoli into blood vessel.
compared to the control rats (p > 0.05). The level of CECs was significantly increased in diabetic rats compared to nondiabetic rats (p < 0.05). The level of CECs was significantly increased in diabetic rats exposed to coal dust at doses of 6.25 mg/m3 and 12.5 mg/m3 (p < 0.05) compared to that in diabetic rats not exposed to coal dust.
Effect of PM10 coal dust on foam cells counts in aorta The foam cells counts were significantly increased in the aorta of nondiabetic rats exposed to coal dust at doses of
Figure 2 Thiobarbituric acid reactive substance (TBARS) levels in nondiabetic and diabetic rats exposed to coal dust. Values are presented as mean standard deviation. a p < 0.05; in comparison with control group. b p < 0.05; in comparison with normal rats exposed to 6.25 mg/m3 coal dust. c p < 0.05; in comparison with normal rats exposed to 12.5 mg/m3 coal dust. d p < 0.05; in comparison with normal rats exposed to 25 mg/m3 coal dust. e p < 0.05; in comparison with diabetic rats. f p < 0.05; in comparison with diabetic rats exposed to 6.25 mg/m3 coal dust.
6.25 mg/m3 and 25 mg/m3 over that in the control rats (p < 0.05). Foam cells counts were significantly increased in aorta of diabetic rats exposed to coal dust at doses of 6.25 mg/m3 and 12.5 mg/m3 compared to control diabetic rats (p < 0.05), as shown in Fig. 4.
Effect of PM10 coal dust on foam cells counts in tail artery Foam cells count was significantly increased in tail artery of nondiabetic rats exposed to coal dust at dose of 25 mg/m3 compared to control rats (p < 0.05). The foam cells count was not significantly different in the tail artery of diabetic
Figure 3 Circulating endothelial cells (CECs) numbers in nondiabetic and diabetic rats exposed to coal dust. Values are presented as mean standard deviation. a p < 0.05; in comparison with control group. b p < 0.05; in comparison with normal rats exposed to 6.25 mg/m3 coal dust. c p < 0.05; in comparison with normal rats exposed to 12.5 mg/m3 coal dust. d p < 0.05; in comparison with diabetic rats.
Please cite this article in press as: Setiawan B, et al., Subchronic inhalation of particulate matter 10 coal dust induces atherosclerosis in the aorta of diabetic and nondiabetic rats, Biomarkers and Genomic Medicine (2014), http://dx.doi.org/10.1016/j.bgm.2014.03.002
+
MODEL
Coal dust induces atherosclerosis in aorta
Figure 4 Foam cells counts in aorta (blue bar) and tail artery (red bar) of nondiabetic and diabetic rats exposed to coal dust. Values are presented as mean standard deviation. a p < 0.05; in comparison with control group. b p < 0.05; in comparison with normal rats exposed to 6.25 mg/m3 coal dust. c p < 0.05; in comparison with normal rats exposed to 12.5 mg/m3 coal dust. d p < 0.05; in comparison with normal rats exposed to 25 mg/m3 coal dust. e p < 0.05; in comparison with diabetic rats. f p < 0.05; in comparison with diabetic rats exposed to 6.25 mg/m3 coal dust. g p < 0.05; in comparison with diabetic rats exposed to 12.5 mg/m3 coal dust.
rats exposed to coal dust compared to that in nonexposed diabetic rats (p > 0.05), as shown in Fig. 4.
Discussion Prior to exposure, we characterized coal dust using scanning electron microscopy, which demonstrated a diameter <10 mm at least in one dimension, in morphology of singlet or aggregate particles, with crystal size 177 nm. We arrived at two main findings. The first finding concerns the accumulation of PM10 coal dust in pulmonary blood vessel. The second is the effect of PM10 coal dust on foam cell formation as a vascular complication of diabetes mellitus. The accumulation of coal dust in lung blood vessel indicates the potential translocation of the dust from the alveolar into the circulatory system. The mechanism of translocation involves the increase in the lung epithelium permeability induced by oxidative stress. Furthermore, this process opens up the access of particles into the interstitial compartment and they continue to pass into the endothelial cells and the blood, and then they are circulated to several organs.24 Coal dust in nanoparticles is able to translocate from the lungs into the circulatory system directly. Although only very small doses of coal dust nanoparticles enter the circulation (1%), the effect in the targeted organs is caused by large surface particles and the retention time.25 TBARS is the product of lipid peroxidation as an oxidative stress marker. We found an increase in oxidative stress in all of the groups, when comparing them with the control group of nondiabetic rats. Oxidative stress in diabetic rats with or without coal dust inhalation was significantly higher than that in the nondiabetic rats. In nondiabetic rats, the inhalation of coal dust increased oxidative stress in a dose dependent manner compared to the control group (p < 0.05). In diabetic rats, coal dust inhalation significantly increased oxidative stress when compared to the control group at doses of 12.5 mg/m3 and 25 mg/m3 (p < 0.05). The coal dust used in this study contained
5 several metal redox properties, such as iron (29.3 0.1%), titanium (6.31 0.19%), chromium (0.48 0.04%), nickel (0.41 0.00%), vanadium (0.20 0.02%), and manganese (0.15 0.09%). This active metal content induces lipid peroxidation by a mechanism that is both hydroxyl radical dependent and nonhydroxyl radical dependent.26,27 In addition, hyperglycemia in diabetes mellitus also induces oxidative stress via several mechanisms, such as nonenzymatic glycation, the polyol pathway, and glucose autooxidation.28 There is accumulation of oxidative stress in diabetic rats with the inhalation of coal dust. CECs are mature cells detached from the intimal monolayer of the vascular wall. The count of these cells circulating in the peripheral blood is a biomarker of vascular damage. Several mechanisms can be related to endothelial detachment, including apoptosis, mechanical damage of cells, and protein lysis of the subendothelial matrix.29,30 Depending on the stress level, a severe degree of oxidative stress induces apoptosis in endothelial cells.14 We found a significant increase in CECs in diabetic groups compared to nondiabetic groups without coal dust exposure. This finding indicates that hyperglycemia increases endothelial damage and detachment. Coal dust increases endothelial damage in diabetic rats (at doses of 6.25 mg/m3 and 12.5 mg/m3) over that in control diabetic rats. This finding indicates that active metals in coal dust can damage endothelial cells may be due to apoptosis, mechanical damage of cells, protein lysis of the subendothelial matrix, or oxidative stress. At dose of 25 mg/m3, coal dust decreases circulating endothelial cells in diabetic rats to reach the level of control diabetic rats. This finding indicates that coal dust at this dose inhibits endothelial detachment in diabetic rats. The mechanism may be due to an inhibition of aggressive factor in diabetic rats. Free radicals from coal dust will react with the free radicals from hyperglycemia to induce the termination (neutralization) reaction. Our previous studies showed that coal dust exposure tended to decrease endothelial cells damage in rats fed a high-cholesterol diet.31 Foam cells are a histologic marker of early atherosclerotic lesions. In this study, we assessed the foam cell formations in large and small arteries of rats. In nondiabetic rats, coal dust inhalation significantly increased the foam cells count in the aorta at doses of 6.25 mg/m3 and 25 mg/m3 compared to the control group (p < 0.05). Moreover, when nondiabetic rats are exposed to 25 mg/m3 coal dust, foam cells increased in the tail artery compared to the control and lower exposure dose. These findings indicate that active metals in coal dust initiate an early lesion of atherosclerosis in large and small vessels of nondiabetic rats. Increased oxidation of LDL-cholesterol is a key feature of foam cells, and transition metals can increase both direct LDL-cholesterol oxidation and LDL oxidation by monocytes.32,33 Also, previous study have proved that coal dust increases systemic inflammation.13 In diabetic rats, inhalation of coal dust significantly increased foam cells counts in aorta at doses of 6.25 mg/m3 and 12.5 mg/m3 compared to the control group (p < 0.05), but this did not reach statistically significance in the small artery. Actually, the concentration of collagen and elastin in the aorta is inversely related to that in the tail artery in rats. Collagen is lower in the aorta, but higher in the tail
Please cite this article in press as: Setiawan B, et al., Subchronic inhalation of particulate matter 10 coal dust induces atherosclerosis in the aorta of diabetic and nondiabetic rats, Biomarkers and Genomic Medicine (2014), http://dx.doi.org/10.1016/j.bgm.2014.03.002
+
MODEL
6 artery.34 Collagen induces progression of atherosclerotic lesion by serving as a depot of proatherogenic molecules.35 Elastin is an extracellular protein with lower turnover ratesdalmost 40 years.36 The total alkaline-insoluble aorta elastin content is significantly reduced in diabetic compared to control rats.37 The combination of collagen and elastin content in the aorta, oxidative stress level related to coal dust exposure (at doses of 6.25 mg/m3 and 12.5 mg/m3), and hyperglycemia, may be the reason for proatherogenic molecules deposition forming foam cells in the aorta of diabetic rats.38 At the highest dose, coal dust inhibited foam cells formation in the aorta or tail artery. Our previous study showed that coal dust decreases cholesterol and LDL-cholesterol levels at an exposure dose of 25 mg/m3, and this effect may contribute to this finding.39 In conclusion, our study found that subchronic inhalation of PM10 coal dust induces atherosclerosis through oxidative stress and endothelial damage in the aorta of nondiabetic and diabetic rats.
Conflicts of interest The authors declare that there is no conflicts of interests regarding the publication of this article.
Acknowledgments The authors are grateful to PT. Carsurin Banjarmasin, South Kalimantan for providing coal dust in diameter <70 mm. The authors also thank all technician in the Department of Pharmacology and Laboratory of Biomedicine (Faculty of Medicine, Brawijaya University, Malang, East Java, Indonesia) for helping in the procedure of coal dust exposure and analyses of the biological marker.
References 1. Huang X. Iron, oxidative stress, and cell signalling in the pathogenesis of coal worker’s pneumoconiosis, silicosis, and asbestosis. Am J Biomed Sci. 2001;3:95e106. 2. Hendryx M, Zullig KJ. Higher coronary artery disease and heart attack morbidity in Appalachian coal mining regions. Preventive Med. 2009;49:355e359. 3. Bonnefont-Rousselot D. Glucose and reactive oxygen species. Curr Opinion Clin Nutr Metab Care. 2002;5:561e568. 4. Wu T, McGrath KCY, Death AK. Cardiovascular disease in diabetic nephropathy patients: cell adhesion molecules as potential markers. Vasc Health Risk Manag. 2005;1:309e316. 5. Rajagopalan S, Brook RD. Air pollution and type 2 of diabetes. Diabetes. 2012;61:3037e3045. 6. Chuang KJ, Yan YH, Chiu SY, et al. Long-term air pollution exposure and risk factors for cardiovascular diseases among the elderly in Taiwan. Occup Environ Med. 2011;68:64e68. 7. Dalal NS, Newman J, Pack D, et al. Hydroxyl radical generation by coal mine dust: possible implication to coal workers pneumoconiosis. Free Radic Biol Med. 1995;18:1e20. 8. Zhang Q, Dai J, Ali A, et al. Roles of bioavailable iron and calcium in coal dust-induced oxidative stress: Possible implications in coal workers’ lung disease. Free Rad Res. 2002;36: 285e294.
B. Setiawan et al. 9. Zhang Q, Huang X. Induction of ferritin and lipid peroxidation by coal samples with different prevalence of coal workers’ pneumoconiosis: role of iron in the coals. Am J Ind Med. 2002; 42:171e179. 10. Armutcu F, Gun BD, Altin R, et al. Examination of lung toxicity, oxidant/antioxidant status and effect of erdosteine in rats kept in coal mine ambience. Environ Toxicol Pharmacol. 2007; 24:106e113. 11. Pinho RA, Silveira PCL, Piazza M, et al. Regular physical exercises decrease the oxidant pulmonary stress in rats after acute exposure to mineral coal. Rev Bras Med Esporte. 2004;12: 71ee74e. 12. Avila Ju ´nior SA, Possamai FP, Budni P, et al. Occupational airborne contamination in South Brazil: oxidative stress detected in blood of coal miners. Ecotoxicol. 2009;18:1150e1157. 13. Ulker OC, Yucesoy B, Demir O, et al. Serum and BAL cytokine and antioxidant enzyme level at different stages of pneumoconiosis in coal workers. Hum Exp Toxicol. 2008;27:871e877. 14. Aoki M, Nata T, Morishita R, et al. Endothelial apoptosis induced oxidative stress through activation of NFkB: antiapoptotic effect of antioxidant agents on endothelial cells. Hypertension. 2001;38:48e55. 15. Boos CK, Lip GYH, Blann AD. Circulating endothelial cells in cardiovascular disease. J Am Col Cardiol. 2006;48:1538e1547. 16. Lane HA, Smith JC, Davies SJ. Noninvasive assessment of preclinical atherosclerosis. Vasc Health Risk Manag. 2006;2: 19e36. 17. Kania N, Mayangsari E, Setiawan B, et al. The effects of Eucheuma cottonii on signaling pathway inducing mucin synthesis in rats lungs chronically exposed to particulate matter 10 (PM10) coal dust. J Toxicol. 2013;2013:528146. 18. Cheng D, Liang B, Li Y. Antihyperglycemic effect of Ginkgo biloba extract in streptozotocin-induced diabetes in rats. BioMed Res Int. 2013;2013:162724. 19. Gurel A, Armutcu F, Damatoglu S, et al. Evaluation of erythrocyte Naþ, Kþ, -ATPase and superoxide dismutase activities and malondialdehyde level alteration in coal miners. Eur J Gen Med. 2004;1:22e28. 20. Noor Z, Setiawan B. Subchronic inhaled particulate matter coal dust changes bone mesostructure, mineral element and turn over markers in rats. J Exp Integr Med. 2013;3:153e158. 21. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95: 351e358. 22. Hladovec B, Rossman P. Circulating endothelial cell isolated together with platelets and experimental modification of their counts in rats. Thrombos Res. 1973;3:665e674. 23. Kania N, Setiawan B, Widjajanto E, et al. Peroxidative index as novel marker of hydrogen peroxide involvement in lipid peroxidation from coal dust exposure. Oxid Antioxid Med Sci. 2012;1:209e215. 24. Donaldson K, Stone V, Seaton A, et al. Ambient particle inhalation and the cardiovascular system: potential mechanisms. Environ Health Perspect. 2001;109:523e527. 25. Delfino RJ, Staimer N, Vaziri ND. Air pollution and circulating biomarkers of oxidative stress. Air Quality Atmos Health. 2011; 4:37e52. 26. Broeckaert F, Buchet JP, Delos M, et al. Coal fly ash- and copper smelter dust-induced modulation of ex vivo production of tumor necrosis factor-alpha by murine macrophages: effects of metals and overload. J Toxicol Environ. 1999;56:343e360. 27. Valko M, Rhode CJ, Moncol J, et al. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact. 2006;160:1e40. 28. Mokini S, Marcovecchio ML, Chiarelli F. Molecular pathology of oxidative stress in diabetic angiopathy: role of mitochondrial and cellular pathways. Diabetes Res Clin Pract. 2010;87: 313e321.
Please cite this article in press as: Setiawan B, et al., Subchronic inhalation of particulate matter 10 coal dust induces atherosclerosis in the aorta of diabetic and nondiabetic rats, Biomarkers and Genomic Medicine (2014), http://dx.doi.org/10.1016/j.bgm.2014.03.002
+
MODEL
Coal dust induces atherosclerosis in aorta 29. Kondo S, Ueno H, Hashimoto J, et al. Circulating endothelial cells and other angiogenesis factors in pancreatic carcinoma patients receiving gemcitabine chemotherapy. BMC Cancer. 2012;12:268. 30. Elshal M, Abdelaziz A, Abbas A, et al. Quantification of circulating endothelial cells in peripheral blood of systemic lupus erythematosus patients: a simple and reproducible method of assesing endothelial injury and repair. Nephrol Dial Transplant. 2009;24:1495e1499. 31. Setiawan B, Darsuni A, Muttaqien F, et al. The effects of combined particulate matter 10 coal dust exposure and highcholesterol diet on lipid profiles, endothelial damage, and hematopoietic stem cells in rats. J Exp Integr Med. 2013;3: 219e223. 32. Xing X, Baffic J, Sparrow CP. LDL oxidation by activated monocytes: characterization of oxidized LDL and requirement for transition metals ions. J Lipid Res. 1998;39:2201e2208. 33. Retsky KL, Chen K, Zeind J, et al. Inhibition of copper-induced LDL oxidation by vitamin C is associated with decreased
7
34. 35. 36.
37.
38.
39.
copper-binding to LDL and 2-oxo-histidine formation. Free Rad Biol Med. 1999;26:90e98. Cox RH. Time course of artery wall changes with DOCA plus salt hypertension in the rat. Hypertension. 1982;4:27e38. Rekhter MD. Collagen synthesis in atherosclerosis: too much and not enough. Cardiovas Res. 1999;41:376e384. Yamamoto Y, Sakata N, Meng J, et al. Possible involvement of glycoxidation and lipid peroxidation of elastin in atherogenesis in haemodialysis patients. Nephrol Dial Transplant. 2002;17: 630e636. Kwan CY, Wang RR, Beazley JS, et al. Alterations of elastin and elastase-like activities in aortae of diabetic rats. Biochim Biophys Acta. 1988;967:322e325. Francis AA, Pierce GN. An integrated approach for the mechanisms responsible for atherosclerotic plaque regression. Exp Clin Cardiol. 2011;16:77e86. Setiawan B, Darsuni A, Muttaqien F, et al. Cholesterol lowering effect of subchronic inhalation particulate matter 10 coal dust on rats. Med-Sci. 2013;2:500e511.
Please cite this article in press as: Setiawan B, et al., Subchronic inhalation of particulate matter 10 coal dust induces atherosclerosis in the aorta of diabetic and nondiabetic rats, Biomarkers and Genomic Medicine (2014), http://dx.doi.org/10.1016/j.bgm.2014.03.002